This is a new R01 proposal entitled "Structures of Eukaryotic K Channels and Mistic Action" will focus on two main goals: 1) understanding how Mistic works to permit insertion and topogenesis of eukaryotic Integral Membrane (IM) proteins into E. coli membrane, and 2) three dimensional structures of eukaryotic voltage-gated K (Kv) channels by x-ray crystallography methods. Our proposed study will aim to expand scope and direction to a newer level by focusing on molecular working of 6 transmembrane (TM)-helix voltage-gated K channels. In the first Aim, we propose to study action mechanisms of Mistic, a novel chaperone protein that permits autonomous insertion of eukaryotic IM proteins into E coli membrane. We will analyze protein interaction with a model cargo protein, KvPae in association with Mistic. This work will address the structural basis of membrane topogenesis of IM proteins and their conformational flexibility. In the second Aim, we propose to determine crystal structure of eukaryotic Kv channels. We will establish a FLAG-based antibody in combination with FLAG-engineered Kv channel proteins to facilitate their crystallization. Furthermore, we will analyze protein interface between the entire N-terminal domain of eukaryotic Kv channel, a Kv1.1 , and its transmembrane domain. We will analyze the conformational change by NMR spectroscopy to address the structural basis of mechanism of channel inactivation by its own inactivation subdomain and by protein-protein interaction. In summary, we will probe structural changes occurring in the intact isolated eukaryotic Kv channels primarily by a combination of x-ray crystallography and NMR spectroscopy methods. We will learn a great deal about structural mechanisms underlying voltage activation and inactivation of eukaryotic Kv channels.